RESUMO
DFNA16 is a form of autosomal dominant non-syndromic hearing loss (ADNSHL) characterized by fluctuating progressive hearing impairment. Earlier, we mapped the deafness-causing gene to chromosome 2q23-24.3. In this paper, we describe fine mapping results using additional markers tightly linked to the DFNA16 candidate region. Critical recombinants at markers D2S354 and D2S124 define a 3.5-cM interval that contains the DFNA16 gene. Positional candidate genes include two members of the voltage-gated sodium channel family, the type 2 alpha subunit (SCN2A) and the type 3 alpha subunit (SCN3A). After showing that SCN2A is expressed in human fetal cochlea, we determined its genomic structure to facilitate mutation screening in our DFNA16 kindred. We also determined the genomic structure of SCN3A. These two genes are oriented head-to-head, with their 5' ends separated by approximately 40 kb; their homology is 82% at the nucleotide level, and 85% for identities and 90% for positives at the amino acid level. They share similar genomic structures and have alternative splice isoforms that are developmentally regulated and highly conserved between species. Although no DFNA16-causing mutations were found in either gene, haplotype analysis with polymorphic markers in SCN2A introns further narrowed the candidate gene interval to the region flanked by D2S354 and STS SHGC-82894.
Assuntos
Genes/genética , Proteínas do Tecido Nervoso/genética , Canais de Sódio/genética , Processamento Alternativo , Sequência de Bases , Cromossomos Humanos Par 2/genética , Mapeamento de Sequências Contíguas , DNA/química , DNA/genética , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Surdez/genética , Éxons , Saúde da Família , Feminino , Humanos , Íntrons , Masculino , Repetições de Microssatélites , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2 , Canal de Sódio Disparado por Voltagem NAV1.3 , Linhagem , Mapeamento Físico do Cromossomo , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita Simples , Subunidades Proteicas , Análise de Sequência de DNARESUMO
The prevalence of penicillin non-susceptible Streptococcus pneumoniae (PNSSP) is increasing among isolates from acute otitis media (AOM). Repeated episodes of antibiotic exposure are a well-known risk factor for the isolation of PNSSP although otitis-prone or recurrent AOM cases frequently require repeated courses of antibiotic treatment. In order to evaluate the chronological alteration of S. pneumoniae during recurrences of AOM, strains of S. pneumoniae were isolated from 11 patients, each of whom had experienced 2-4 episodes of AOM, were examined. Every bacterial specimen obtained from a single episode of recurrent AOM was examined by PCR-based penicillin-binding protein (PBP) assay, serotyping, and amplified fragment length polymorphism (AFLP), then compared to other samples from the same case. Two cases (18.2%) showed strain diversity during repeated antibiotic treatments by serotyping or PBP-assay. By AFLP analysis, 6 cases (54.5%) demonstrated heterogeneous strains during recurrent AOM. Clonal survivors of previous episodes of AOM were not always the cause of subsequent episodes of AOM, even in otitis-prone cases.
Assuntos
Otite Média/tratamento farmacológico , Otite Média/genética , Resistência às Penicilinas , Polimorfismo Genético , Streptococcus pneumoniae/genética , Doença Aguda , Variação Genética , Humanos , Mutação , Reação em Cadeia da Polimerase , Ligação Proteica , Análise de Sequência de DNARESUMO
The spread of penicillin non-susceptible Streptococcus pneumoniae (PNSSP) is an emerging problem for the treatment of acute otitis media (AOM). Attendance of children at day care centers, as well as the spread of PNSSP, is a risk factor for AOM. The status of the spread of PNSSP during the acute infection phase of AOM has not been evaluated. We examined the clonality of samples from seven children in a day care center who simultaneously developed AOM caused by PNSSP. The seven isolates from the children, and six control samples were grouped by serotyping, by determining resistance to antimicrobial agents, and by genotyping, carried out by sequencer-based random amplified polymorphic DNA (RAPD), and validated by bootstrap analysis. There was no evidence to indicate the direct dissemination of PNSSP among these patients in the day care center, although the simultaneous occurrence of PNSSP AOM had initially suggested a clonal outbreak. The possible presence of a common ancestral strain suggested the importance of surveillance during the carrier state. The result of RAPD genotyping was highly reproducible, as validated by the high bootstrap score. The use of an automated sequencer, in combination with a careful choice of primers, and commercially established kits, played a significant role in the reproducibility of the studies.